The present invention is directed to the separation of particles, such as liposomes and lipid particles, according to size using tangential flow filtration. The filtration method as disclosed permits the large scale separation of these particles into select size ranges, the size determined by the pore size of the filter employed. Any of the known tangential flow filtration devices and materials, such as the hollow fiber or tube, or the flat or pleated sheets or films, may be used. Other devices employing tangential flow filtration may also be used.
In the present invention, the terms "tangential flow filtration" and "cross flow filtration" are used interchangably, and are defined as the separation of suspended solids from aqueous or organic fluids or fluid mixture by passing or circulating a sample feed parallel or tangential to the membrane surface, with an effluent of concentrated solids continuing to flow tangential to the membrane. The pore size of the filter determines which particles will be removed in the filtrate, and those retained in the feed (retentate). For example, a sample feed stock passed through a tangential flow filtration device having a 5.0 um pore size filter allows passage of particles less than 5.0 um to pass into the filtrate. Particles larger than 5.0 um remain in the retentate.
Unlike traditional filtration processes, including those employing extrusion and ceramic filtration devices (see Martin et al., U.S. Pat. No. 4,752,425, issued Jun. 21, 1988, and Martin et al., U.S. Pat. No. 4,737,323, issued Apr. 12, 1988), the instant procedure prevents a filter cake build-up on the filter surface. Also, there is no "dead-end" extrusion of larger particles due to pressure, as the liquid is caused to flow across a membrane surface. The flow rate of the liquid is therefore maintained as it is passed over the membrane.
The present invention is directed towards the separation of particles according to size using the tangential flow filtration technique, and is specifically directed towards the size separation of particles such as liposomes and lipid particles. Liposomes and lipid particles made by any method in the art may be separated according to this technique.
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient towards the center of the bilayer while the hydrophilic "heads" orient towards the aqueous phase.
The original liposome preparation of Bangham et al. (J. Mol. Biol., 1965, 12:238-252 involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles (SUVs) described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638), and large unilamellar vesicles.
Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Publication No. 87/00238, Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure through a membrane filter. Vesicles may also be made by an extrusion technique through a 200 nm filter; such vesicles are known as VET.sub.200 s.
Another class of liposomes that may be used are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Pat. No. 4,558,579 to Fountain, et al., and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies".
Other techniques that are used to prepare vesicles include those that form reverse-phase evaporation vesicles (REVs), Papahadjopoulos et al., U.S. Pat. No. 4,235,871, issued Nov. 25, 1980.
A variety of sterols and their water soluble derivatives have been used to form liposomes; see specifically Janoff et al., PCT Publication No. 85/04578, Oct. 24, 1985, entitled "Steroidal Liposomes." Mayhew et al., PCT Publication No. 85/00968, Mar. 14, 1985, described a method for reducing the toxicity of drugs by encapsulating them in liposomes comprising alpha-tocopherol and certain derivatives thereof. Also, a variety of tocopherols and their water soluble derivatives have been used to form liposomes, see Janoff et al., PCT Publication No. 87/02219, Apr. 23, 1987, entitled "Alpha Tocopherol-Based Vesicles."
In a liposome-drug delivery system, a bioactive agent such as a drug is entrapped in or associated with the liposome and then administered to the patient to be treated. For example, see Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schnieder, U.S. Pat. No. 4,114,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.
In the present invention, lipid particles such as those disclosed in commonly owned copending applications, Janoff et al. U.S. patent applications Ser. No. 07/022,157, filed Mar. 5, 1987 and now abandoned, U.S. patent applications Ser. No. 07/069,908, filed U.S. Pat. No. 5,616,334, the relevant portions of which are incorporated herein similar to those for making liposomes, and have lower toxicities than the drugs when administered in their free forms. Such complexes comprise drug in a relatively high mole ratio with one or more lipids. Additionally, liposomes formed using a transmembrane pH gradient according to the methods of copending U.S. patent application Ser. No. 06/749,161, filed Jun. 26, 1985, Bally et al., entitled "Encapsulation of according to the methods of U.S. Pat. Nos. 5,077,056 and 5,616,341, both of which herein are to the invention of Bally et al. mentioned above, liposomes are loaded with ionizable bioactive agents wherein a transmembrane pH gradient is formed across the bilayers of the liposomes, and the agent is loaded by means of this gradient. The ion gradient is generated by creating a concentration gradient for one or more charged species (for example, H.sup.+ ions) across the liposome membranes. Such gradients then drive the accumulation of ionizable bioactive agents, for example, prostaglandins, or antineoplastic agents such as doxorubicin, vincristine, epirubicin, or daunorubicin into the liposomes.
More specifically, to create the concentration gradient, liposomes are prepared in the presence of a first aqueous medium, such medium being both entrapped by and surrounding the liposomes. The external medium of these liposomes is then adjusted to a more acidic or basic pH, such as by exchanging the surrounding medium. Such a process creates the transmembrane concentration gradient. If the second external medium contains an ionizable bioactive agent such as an ionizable antineoplastic agent, the H.sup.+ gradient will partition the drug into the liposomes such that the free vesicle-associated bioactive agent ratios reflect H.sup.+ ! in/H.sup.+ ! out ratios. As disclosed in Mayer et al., identified above, antineoplastic agents such as doxorubicin, daunorubicin, epirubicin, and vincristine may be accumulated in liposomes at high drug:lipid ratios by this method, also referred to as a "remote loading" method. Such liposomes may be passed across the tangential flow filtration device and separated according to their size.
As disclosed hereinabove, when used in the process of separating liposomes or lipid particles, tangential flow filtration may be used with liposomes or lipid particles made by any of the methods known. In addition to the above-named methods, both the liposomes or lipid particles may be formed by additional or alternative processes such as shearing or sonication. In one aspect of the invention, they are formed by a homogenization technique, such as those employing homogenization, colloid milling, or size reduction extrusion process devices. When any of these devices are employed, the homogenization device may be connected directly to (in series with) the tangential flow filtration device. Alternatively, the homogenization process may be carried out independently of the filtration device.
The use of tangential flow or cross flow filtration for the gross separation of materials is known. Marinaccio et al., (WO 85/03011, published Jul. 18, 1985) disclose the process for use in the separation of biological liquids such as blood components for plasmapheresis. In this process, blood is passed tangentially to (i.e., across) an organic polymeric microporous filter membrane, and particulate matter is removed. In another use, tangential flow filtration has been disclosed for the filtration of beer (Shackleton, EP 0,208,450, published Jan. 14, 1987) specifically for the removal of particulates such as yeast cells and other suspended solids. Kothe et al., (U.S. Pat. No. 4,644,056, issued Feb. 17, 1987) disclose the use of this process in the purification of immunoglobulins from milk or colostrum, and Castino (U.S. Pat. No. 4,420,398, issued Dec. 13, 1983) describes its use in the separation of antiviral substances such as interferons from broths containing these substances as well as viral particles and cells.
Tangential flow filtration units have been employed in the concentration of cells suspended in culture media. The size of the membrane used has been chosen with regard to efficiency and speed of processing and separating the cells. Radlett (1972, J. Appl. Chem. Biotechnol., 22:495) proposes tangential flow filtration as an alternative to the more commonly used cell separation methods such as centrifugation and conventional filtration.
Similarly, the technique has been used in the separation of bacterial enzymes from cell debris (Quirk et al., 1984, Enzyme Microb. Technol., 6(5):201). Using this technique, Quirk et al. were able to isolate enzyme in higher yields and in less time than using the conventional technique of centrifugation. The use of tangential flow filtration for several applications in the pharmaceutical field has been reviewed by Genovesi (1983, J. Parenter. Aci. Technol., 37(3):81), including the filtration of sterile water for injection, clarification of a solvent system, and filtration of enzymes from broths and bacterial cultures.
The control of particle size in a population is difficult and generally has not been successful. The present invention of the use of tangential flow filtration in the separation of liposomes or lipid particles according to size is a commercially important process. The use of filters of selected sizes, and further, the sequential use or serial attachment of filters of different sizes (i.e., a filtering system) is disclosed for the separation of particles to obtain particles of a specifically desired size range.
There are problems associated with previous attempts to select liposomes according to size. For example, Huang (1969, Biochemistry, 8:344) describe a multi-step technique for the production of small unilamellar vesicles (SUVs) involving sonication, centrifugation, filtration of the population through a 0.1 um dead-end filter, and finally subjecting the filtrate to molecular sieve chromatography on a Sepharose 4B column to remove the large liposomes. Barenholz et al., (1977, Biochemistry, 16:2806) developed a technique employing sonication, centrifugation to remove large liposomes, followed by high speed centrifugation for 1 to 4 hours. This process similarly produced SUVs. Watts et al. (1978, Biochemistry, 17:1792) prepared a homogenous SUV population of dimyristoylphosphatidylcholine (DMPC) by sonication followed by centrifugation at 105,000.times.g.
In addition to the efforts directed at obtaining homogenous populations of SUVs, numerous attempts have been made to obtain homogenous populations of larger liposomes, i.e., MLVs. The majority of these efforts have involved the use of a series of membrane filters in an extrusion process. Such extrusion techniques involve the sequential extrusion of MLVs through filters having various pore sizes (Olson, et al., 1979, Biochim. Biophys. Acta., 557:9, and Schullery et al., 1973, Chem. Phys. Lipids, 12:75). Such a process forms a mixed population of MLVs and SUVs. These liposomes were found not to possess a homogenous, unimodal distribution with regard to size, but were in fact contaminated by liposomes of much larger and smaller size. A unimodal distribution is one in which the chi square value of the Gaussian distribution of the particle size is less than or equal to 2.0. In addition, these techniques are liposome formation techniques, as opposed to the present invention of selection of liposomes of defined size ranges from a heterogenously-sized population.
Martin et al. (U.S. Pat. No. 4,752,425, issued Jun. 21, 1988) have disclosed methods for forming liposomes of high encapsulation efficiency employing the infusion of lipids containing solvent and drug, into an aqueous solution. The method further involves the extrusion of the resulting liposomes through ceramic filters. During the infusing step, the suspensions can be diafiltered to form a filtrate of liposomes of 0.1 um and less.
There remains a difficulty in the art of obtaining a homogenous population of liposomes having a defined upper and lower size range. The present invention solves this problem by allowing selection of liposomes of a homogeneous, defined size distribution from a heterogenously-sized population. The use of filters of selected sizes is disclosed for the separation of particles of defined size. A homogeneous distribution of particles is a population of particles having a known, well-defined size distribution with essentially no particles above a certain size and essentially no particles below a certain size. As used in the present invention, the term "essentially" shall be understood to mean no more than about 10% of the particles, and preferably no more than 5% of the particles are of sizes above or below the defined size as determined by the tangential flow filter sizes. In the art, such a distribution, for example, a difference between essentially the largest and essentially the smallest particle sizes of 3, 4, 5, 10, or 100 microns, are generally not known, yet are routinely achievable in the present invention. In certain cases the resulting homogeneous distribution of liposomes or lipid particles is unimodal.
The degree to which particle size homogeneity can be obtained is influenced by the physical and chemical characteristics of the sample and the filtration conditions. For example, the viscosity and composition (charge) of the sample or the suspending solution, and the pore size and composition of the filters (thickness; presence of an asymmetric skin on the filter; charge, which influences the binding or repelling of the sample to the filter; etc.) also determine the efficiency of the filtration process and the homogeniety of the final product.
In the liposome or lipid particle sizing application, such filters may be attached downstream (in series) from a homogenization or milling apparatus; such apparatus outputs sample into the filter or filtering system (two filters, enabling the defining of particles with an upper and lower size cut-off). Alternatively, the homogenization device may be used independently, and the resulting homogenized material applied to the filtration device manually or in a separate step. In either case, the resulting filtrate (or retentate, depending on the desired product) is collected as final product. The material not passing through the filter(s) (the retentate) due to its large size may then be discarded, or alternatively recycled back through the homogenization or milling apparatus for re-sizing, and then back through the filtering device. The total yield of filtrate generally increases following each complete cycle.
Alternatively, two or more tangential flow filtration devices may be connected in parallel with the homogenization or milling apparatus. In such case the filtration devices may contain filters of different sizes, allowing separation of the same feed sample into products of differing size. In this case, if the sample is liposomes, lipid particles, or another material for sizing, the retentate may be cycled back to the homogenization or milling apparatus, to undergo further sizing adjustment.
When particles of a discrete size having both upper and lower size limits are desired, the homogenization or milling apparatus may be connected to at least two filtration devices, positioned in series, one having a filter pore size of the upper particle size limit desired, and the second having a filter pore size of the lower particle size limit. As the sample passes through the first filter, particles that are below the limit of the pore size pass through into the filtrate. The retentate may then be recycled back through the homogenization or milling apparatus for further size adjustment. The filtrate is then cycled through the next filter having the lower limit pore size. The particles smaller than this size are passed into the filtrate, and the filtrate may be discarded. The retentate thus contains all particles between the upper and lower defined size limits.
Although this technique is useful in the separation of small batches of sample feed, it is particularly useful in the large scale size separation of liposomes or lipid particles, as such separation may be easily accomplished with large volumes of material without the problems normally associated with the filtration of lipids. Such problems arise when liposomes or lipid particles are subjected to traditional "dead-end" filtration processes, as some liposomes or particles may be deformed by the pressures needed to pass them across the filter. When these particles are deformed, they may pass through pore sizes smaller than the actual particle size, and reform on the downstream side of the filter. Thus, the filtrate may be contaminated with particles of sizes outside the desired range. A second problem in the use of traditional filtration for separation of products is the product build-up on the filter surface and the eventual clogging of the membrane pores. The sweep of material tangential to the filter surface, the present technique, prevents this build-up. Additionally, in the lipid applications suggested in the present disclosure, the lower pressures employed by the tangential flow process (typically, lower than 50 psi) are less likely to cause physical damage (i.e. shearing) to the liposomes or lipid particles. Other advantages of the invention over dead-end filtration are the continuous cycling of sample, and the ability to wash out impurities from the retentate. The sample may also be concentrated by removing suspending solution from the sample, thereby resulting in a product of desired potency.
In another aspect of the present invention, the tangential flow filtration device may be used to form liposomes or lipid particles. In such a process, amphipathic materials such as lipids, and bioactive agents suspended in aqueous solutions are caused to contact one another across the membrane surfaces of a tangential flow filter. Controlled pressure delivered by a pump, and shear forces encountered at the membrane surface cause the interaction of the lipid and aqueous components and can be regulated to effect influx of one phase into the other. The control of this biphasic mixing allows the manufacture of liposomes in the defined size range desired, determined by the filter pore size and the pump pressure. More specifically, a solution containing a lipid suspension is caused to contact a first side of a tangential flow filter, while an aqueous solution, which can contain a bioactive agent such as a drug, is infused or injected into the area surrounding a second side of the filter. Pressures delivered to the aqueous side of the filter via a pump cause the passage of the aqueous solution across the filter, through the pores, to the lipid-containing side. Liposomes form at the lipid side of the filter.
In particular, a lipid suspended in an organic solvent, for example, egg phosphatidylcholine in ethanol at about 100-1000 mg/ml is passed in the extracapillary space of a hollow fiber tangential flow filter, for example; and an aqueous solution, such as for example, buffer or a saccharide solution is passed through the lumen of the filter. In response to applied pump pressures, the aqueous solution passes through the pores of the tangential flow filter and forms liposomes in the lipid solution on the extracapillary side. Such a system is a continuous flow system, which allows formation of large volumes of liposomes. In an alternative method, dynamic rotary flow filtration can be employed in a similar technique to form liposomes. Such a filtration process employs a rotary flow filtration unit such as the Benchmark Rotary Filtration unit (Membrex, Inc., Garfield, N.J.). In this method, the lipid suspension is passed through the lumen of the filter, and the aqueous solution passed through the extracapillary space. The flat, cylindrical filter, attached to a rotating shaft, produces vortices, when rotated, that cause the aqueous solution to pass through the filter into the lumen. At the interface between the filter surface and the lumen, the aqueous solution forms liposomes with the lipid circulating in the lumen. This liposome product is then removed from the lumen.
Another aspect of the present invention which also employs tangential flow filtration is the separation of liposomes or lipid particles from solvents or, alternatively, from free (unentrapped or unassociated) drug in the preparation. Such extraliposomal or extralipid particle materials may be removed by their ability, for example, to pass through the membrane pores, while the liposomes or particles remain circulating in the retentate. The use of filter sizes smaller than the desired liposome or particle size permits the passage of these smaller solvent or free drug molecules through the filter pores, while retaining the desired product. Such a use minimizes, or may eliminate the need for exhaustive rotary evaporation or related techniques for solvent removal. It also eliminates the need for chromatographic separation of free particulates such as free drug, from the final preparation. Both of these processes may be simultaneously performed with the size separation function of the instant invention. Alternatively, a liposome or particle population may be exposed to a solvent- or free drug-removal step prior to the size separation (filtration) step. All processes, however, may be performed by the same device, by choosing the appropriate filter size specific to the function desired. The filter size chosen depends on the size of the molecules or particles to be removed.
Still another use for the tangential flow filtration device is in the separation and classification of microcapsules, suspensions, emulsions, and other small particle systems, such as mixture of different cells, according to size.
An additional advantage of the present invention is that the separations can be done aseptically. Aseptically preparing liposome or lipid particles of defined size distribution has been an ongoing problem.